And Levofloxacin Against Intracellular Staphylococcus Aureus Or Listeria Monocytogenes in J774 Macrophages
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Activity of Garenoxacin (BMS284756) and Levofloxacin Against Intracellular Staphylococcus aureus or Listeria monocytogenes in J774 Macrophages Poster #A1176 P.M. Tulkens C. Seral, P.M. Tulkens and F. Van Bambeke Pharmacologie cellulaire et moléculaire UCL 73.70 av. Mounier 73 Pharmacologie Cellulaire et Moléculaire, Université catholique de Louvain - Brussels - Belgium 1200 Brussels - Belgium [email protected] ABSTRACT INTRODUCTION METHODS (cont’d) RESULTS : pharmacokinetics CONCLUSION Background: ! Eradication of intracellular infections requires the use of antibiotics Activity against intracellular Staphylococcus aureus ATCC 25923: ! Kinetics of Accumulation Both quinolones show a concentration - dependent Quinolones are active against a variety of intracellular able to accumulate in eucaryotic cells at sufficiently high Bacteria were opsonized by human serum during 30 min. Cells were intracellular activity. concentrations.1 Fluoroquinolones (zwitterions) accumulate in cells organisms. Yet, little is known about the relationships infected with an inoculum of 0.5 bacteria/macrophage, and washed and could therefore be used against intracellular bacteria.1,2 garenoxacin 5 µg/ml ! Quinolone activity is lower intracellularly than between intrinsic activity (as determined in broth), cell during 1 h with gentamicin 50 µg/ml after 1 h of phagocytosis at 37°C 6 extracellularly, suggesting a marked defeating effect of accumulation, and intracellular activity. to remove non-phagocytosed and non-firmly adherent bacteria. Cells ! Garenoxacin (BMS-284756; formerly known is T-3811) is a novel the intracellular milieu on activity as compared to broth. des-fluoro (6)-quinolone more active against Gram-postive bacteria were then incubated for up to 24 h with garenoxacin or levofloxacin or Methods: 4 levofloxacin 5 µg/ml and intracellular organisms such as chlamydia.3 with gentamicin at its MIC (0.5 µg/ml; control).4 Mφ were infected with serum-opsonized S.a. or untreated ! For L. monocytogenes, the larger cellular accumulation of garenoxacin and its higher activity in broth translate in a L .m. and exposed to increasing concentrations of GAR or Activity against intracellular Listeria monocytogenes: 2 Cellular to Extracellular larger intracellular activity compared to levofloxacin. LVX (in a clinically meaningful range) for 24 h (S.a.) or 5 h AIM OF THE STUDY Bacteria were infected using an inoculum of 5 bacteria/macrophage, Ratio Concentration (Cc/Ce) 0 (L.m.). Activity in broth was measured in parallel. Cell and washed with PBS after 1 h of phagocytosis at 37°C to remove 0 5 10 15 20 25 30 60120 ! For S. aureus, garenoxacin and levofloxacin show a accumulation of GAR and LVX (at equilibrium and ! To study the intracellular activity of garenoxacin compared to non-phagocytosed and non-firmly adherent bacteria. Cells were time (min.) similar intracellular activity, despite the higher levofloxacin in a model of J774 mouse macrophages infected by a 5 recorded in distinct experiments) was approx 6-fold for incubated for 5 h with garenoxacin or levofloxacin. Kinetics of accumulation of garenoxacin (red squares) and levofloxacin (green accumulation level of garenoxacin. GAR and 3-fold for LVX. phagolysosomal bacteria (Staphylococcus aureus) or by a cytosolic circles) in J774 murine macrophages incubated for up to 2 h with and extracellular bacteria (Listeria monocytogenes). Viable bacteria were determined in cell lysates by colony counting concentration of 5 µg/ml. Results are expressed as the mean ± SD of three ! The relationship between activity in broth, cell (CFU). experiments. accumulation and intracellular activity may vary according Activity (change in log CFU compared to time = 0) ! To correlate intracellular activity with antibiotic cellular accumulation. to the type of infection. Drug in Mφ in broth concentration bacteria (mg/L) RESULTS : pharmacodynamics RESULTS : pharmacodynamics GAR LVX GAR LVX S.a. 0 1.50 ± 0.30 2.90 ± 0.10 METHODS control 0.12 -0.92 ± 0.03 -0.65 ± 0.28 -2.77 ± 0.03 2.83 ± 0.00 ACKNOWLEDGEMENTS levofloxacin 5 µg/ml ± ± ± ± levofloxacin extracellular 0.5 -1.11 0.16 -1.07 0.03 -3.45 0.06 -3.19 0.04 garenoxacin 5 µg/ml Materials: levofloxacin intracellular 1 -1.27 ± 0.05 -1.28 ± 0.02 -3.79 ± 0.00 -3.75 ± 0.07 J774 macrophages, a continuous cell line derived from a mouse In Broth In Cells garenoxacin extracellular The authors are very grateful to M.C. Cambier and N. Aguilera for their ± ± ± ± 4 -1.64 0.16 -1.36 0.05 -4.09 0.00 -3.79 0.00 garenoxacin intracellular reticulosarcoma were maintained at 37°C, in a 5 % CO2 atmosphere, 109 109 expert technical assistance. L.m. 0 0.90 ± 0.10 1.23 ± 0.10 in RPMI medium supplemented by 10 % foetal calf serum. 108 108 0.12 0.66 ± 0.02 0.80 ± 0.05 0.46 ± 0.03 1.20 ± 0.2 107 S. aureus 107 0.5 -0.03 ± 0.04 0.76 ± 0.04 0.06 ± 0.01 0.47 ± 0.01 Levofloxacin [potency 99.7 %] was obtained from Aventis Pharma, Activity on S. aureus Activity on L. monocytogenes 106 106 2 1 -0.54 ± 0.05 0.66 ± 0.02 -1.30 ± 0.01 -0.23 ± 0.01 Antony, France; garenoxacin [potency 99.8 %] from Bristol Myers 3 log 5 5 ) 10 10 REFERENCES 4 -1.22 ± 0.06 -0.42 ± 0.09 -1.70 ± 0.02 -1.54 ± 0.02 Squibb, New Brunswick, CT, USA. 0 h 2 4 4 1 10 4 (CFU 10 10 CFU/mg prot. 1 Cellular pharmacokinetics: 3 3 10 10 - CFU 0 1. Tulkens, P. M. 1991. Intracellular distribution and activity of antibiotics. 0 5 h 102 102 24 h Eur. J. Clin. Microbiol. Infect. Dis. 10:100-106. Cells were incubated for up to 24 h at 37°C with 5 µg/ml of the -1 -CFU 9 9 antibiotic. At the end of the incubation period, cells were washed CFU/ml 10 10 2. Carlier, M. B., Scorneaux B., Zenebergh A., Desnottes J.F., and Tulkens P.M . 1990. (CFU -1 Conclusion: 8 8 -2 10 10 10 0 h Cellular uptake, localization and activity of fluoroquinolones in uninfected and 3 times in ice-cold PBS, collected in H2O or glycine 0.1M, pH 3, and ) Both quinolones show a concentration-dependent 7 L.monocytogenes 7 log -3 sonicated 10 sec at 50 W. Cell lysates were then used for measuring 10 10 -2 infected macrophages. J. Antimicrob. Chemother. 26 Suppl B:27-39. intracellular activity, but the data suggest a marked 6 6 -4 the cell protein content and the antibiotic concentration by an 10 10 3. Fung-Tomc J.C., Minassian B., Kolek B., Huczko E., Aleksunes L., Stickle T., 0 0.12 0.25 0.5 1 4 0 0.12 0.25 0.5 1 4 defeating effect of the intracellular milieu on activity as appropriate method (fluorimetric assay for levofloxacin and radiometric 105 105 Washo T., Gradelski E., Valera L., Bonner D.P. 2000. Antibacterial spectrum of a novel 4 4 des-fluoro(6) quinolone, BMS-284756. Antimicrob. Agents Chemother. 44: 3351-3356. compared to broth. For L.m., the larger cellular assay for garenoxacin). The apparent cellular accumulation (Cc/Ce) 10 4 10 extracellular concentration (µg/ml) was calculated by the ratio of the drug cell content (Cc) to the drug 103 103 accumulation of GAR and its higher activity in broth 4. Seral, C., Van Bambeke, F., and Tulkens, P. M. 2003. Quantitative analysis of extracellular concentration (Ce), assuming that 1 mg of protein 102 102 gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin translated in a larger intracellular activity compared to 0 4 8 12 16 20 24 0 4 8 12 16 20 24 corresponds to 5 µl of cellular volume. (LY333328) activities against intracellular Staphylococcus aureus in mouse J774 LVX. This was not observed for S.a., indicating that the time (h) macrophages. Antimicrob.Agents Chemother. 46: 2095-2103. relationship between activity in broth, cell accumulation Bacterial strain and determination of MICs and MBCs: Variation in the number of CFU as compared to the initial inoculum after 24 h (S.a.) or Left panel. Activity of garenoxacin and levofloxacin 5 µg/ml against 5 h (L.m.) of incubation in broth or in infected J774 macrophages with increasing 5. Carryn, S., Van Bambeke, F., Mingeot-Leclercq, M. P., and Tulkens, P. M. 2002. and intracellular activity may vary according to the type of Hemolysin-producing strain EGD of L. monocytogenes (serotype 1/2a) S. aureus (top) or L. monocytogenes (bottom) in MH broth up to 24 h. concentrations of garenoxacin or levofloxacin (0.12, 0.25, 0.5, 1, 4 µg/ml). Comparative intracellular (THP-1 macrophages) and extracellular activities of beta-lactams, azithromycin, gentamicin, and fluoroquinolones against Listeria infection. and S. aureus ATCC25923 were used for all the experiments. MICs 0, control cells (no antibiotic in broth; for macrophages, control includes gentamicin Right panel. Infected J774 macrophages were exposed to garenoxacin and [0.5 µg/ml] throughout the incubation period). monocytogenes at clinically relevant concentrations. Antimicrob. Agents and MBCs were determined following the recommendations of the levofloxacin for 24 hours at an extracellular concentration of 5 µg/ml. NCCLS. Chemother. 46: 2095-2103. 03-112g.